The present invention relates to fluorescent enzymatic substrates of saccharidic nature having a self-cleavable spacer arm functionalized by a fluorophore F and by at least one inhibitor of the fluorescence of F, to the use thereof for preparation of a diagnostic reagent for functional imaging in vivo, and to the diagnostic reagent for functional imaging containing at least one such enzymatic substrate.
Fluorescence is a very widely used technique for detection of enzymatic activities in vitro. It is an inexpensive, fast and generally highly sensitive technique. Numerous enzymes of significant biological importance have substrates in the form of saccharidic derivatives, which can be used in particular to perform enzymatic assays on biological samples (blood, urine, etc.), on cells (fixed or in culture) or even on tissues (tissues of euthanized animals, biopsies).
Applications that seem equally promising are the in vivo applications, especially for fluorescence imaging of small animals. In fact, reporter genes expressing different enzymes such as β-galactosidase (β-gal), β-glucuronidase (β-glu), chloramphenicol, acetyltransferase, luciferase, fluorescent proteins such as “Green Fluorescent Protein” (GFP) are now very widely used in biology to study gene expression (transcription and translation of DNA in proteins), transfection or other biological processes. The reporter genes can act as indicators to demonstrate the introduction and transcription of another gene of interest situated on the same coding part of the DNA. The DNA constructs containing the reporter genes are introduced into the animal to form transgenic animals. For example, the number of transgenic mice already constructed is very large and growing rapidly. In a very large number of cases, the marker gene used is the lacZ gene, which codes for β-gal of E. coli. Another example of an equally used marker gene is the gusA gene, which codes for β-glu of E. coli. As it happens, the substrates of the enzymes expressed by certain of these genes, and in particular by the lacZ and gusA genes, are saccharidic derivatives. It is therefore very important to have saccharidic substrates available in order that the activity of such enzymes can be detected.
Numerous substrates of saccharidic nature already exist for detection of enzymatic activities, such as, for example, the enzymatic activities of β-gal and β-glu. These enzymatic substrates can be, in particular:                substrates for nuclear imaging,        chemiluminescent substrates such as the substrates sold under the trade names Lumi-Gal® 530 by Lumigen Inc. (USA) and Galacton-Star® by Applied Biosystems (USA);        substrates for dielectrophoretic detection,        substrates for MRI,        substrates forming precipitates,        substrates for spectrophotometric assays, including the X-gal substrate (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside), sold, for example, under the trade name BlueTech® by Mirador DNA Design Inc.; and        fluorescent substrates.        
Ideally, these substrates should have the following properties:                fast enzymatic reaction kinetics,        low Michaelis constant,        large difference between the properties of interest of the substrate and those of its product(s) (properties of interest: absorption for a chromogenic substrate, fluorescence for a fluorogenic substrate, etc.).        
The interest in fluorescent substrates compared with the other substrates described hereinabove is their detection sensitivity and the low cost of the instrumentation necessary to use them. In common with MRI, they make it possible under certain conditions to achieve enzymatic detection in vivo.
In general, the fluorescent enzymatic substrates function according to the following principle: a substrate that does not fluoresce in the detection wavelength region produces a product that fluoresces in that same wavelength region when it is brought into the presence of an enzyme whose activity is to be detected and which is specific to the substrate used. It is therefore necessary to find fluorophores whose fluorescence is initially inhibited when they are grafted onto the substrate and can be liberated after reaction with the enzyme whose activity is to be detected. The choice of commercially available fluorophores is therefore limited by this constraint of initial inhibition of the fluorescence when the fluorophore is fixed on the enzymatic substrate.
In vivo, the recent development of optical methods is opening new horizons for functional imaging. It is now possible to follow, in real time and in non-invasive manner, gene expression in animals, especially in the mouse, after anesthesia. Optical imaging offers a certain number of advantages compared with the other functional imaging techniques, such as magnetic resonance imaging (MRI), positron emission tomography (PET) and single photon emission computed tomography (SPECT):                it obviates the handling of radioactive molecules, therefore removing the attendant constraints and risks (radioprotection, waste management, synchrotron source for the PET markers);        it does not necessitate large investments in instrumentation;        it has good sensitivity compared with MRI, in terms of the amount of marker injected.        
Optical imaging makes use of fluorescent enzymatic substrates.
When the presence of enzymatic activity is to be detected in vivo, for example in a small laboratory animal such as the mouse, very few fluorescent molecules are available for this application. In fact, to ensure that the exciting light and the light emitted by the fluorophore can pass through the tissues, it is advisable to use fluorophores that absorb and emit in the near infrared, or in other words at a wavelength between 640 and 900 nm. As it happens, very few molecules that fluoresce in this wavelength region are commercially available at present (largely limited to the cyanines). The double constraint, or in other words the initial inhibition of fluorescence when the fluorophore is fixed on the substrate and the use of a fluorophore that absorbs and emits in the near infrared, is undoubtedly the reason for the lack of fluorescent enzymatic substrates of saccharidic nature in this wavelength region.
In fact, most fluorescent enzymatic substrates of saccharidic nature currently available on the market are not constructed on the basis of fluorophore groups that absorb and emit in the near infrared. For example, it is possible to procure:                substrates based on fluorescein, for detection of β-gal activity, among which there may be mentioned, for example, FDG (fluorescein-di-β-D-galactopyranoside) (excitation 490 nm/emission 514 nm) or one of its derivatives;        substrates based on coumarins or umbelliferones, for detection of β-gal, β-glu or phosphatase activities, such as the substrates MUG (4-methylumbelliferone β-D-galactopyranoside), DiFMUG (6,8-difluoro-4-methylumbelliferyl β-D-galactopyranoside), MUP (4-methylumbelliferone phosphate), DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate) and derivatives (excitation 350-380 nm/emission 450-470 nm); or else        substrates based on resorufin and derivatives, especially for detection of lipase (excitation 570 nm/emission 585).        
Most fluorescent substrates that are commercially available at present function according to the principle represented in Scheme A below:

In this scheme, the fluorophore F is grafted at anomeric position 1 (anomeric bond of β configuration) onto a monosaccharide, β-glucopyranose, to form the enzymatic substrate. This substrate must be weakly fluorescent before the reaction with the enzyme. The fluorophore groups must therefore be chosen in such a way that their fluorescence can be initially inhibited by the monosaccharide. The enzymatic reaction induces cleavage of the anomeric bond and liberates the fluorophore group. When the fluorophore group is distant from the monosaccharide, its fluorescence is no longer inhibited and it can then emit a signal that is detected by means of a spectrofluorimeter. The emitted signal corresponds to the enzymatic activity and, within a certain concentration range, is proportional to the enzyme concentration.
Nevertheless, the substrates functioning according to the principle illustrated in Scheme A exhibit a certain number of disadvantages:                the choice of fluorophore group is limited by the fact that its fluorescence must be capable of being inhibited when it is grafted onto the sugar; not all fluorophore groups have this property, especially in the near infrared region when the capability of detection in vivo is desired (see the foregoing). Thus the commercial fluorogenic substrates always have fluorophores of the same families: coumarins, umbelliferones, fluoresceins, resorufins. In the near infrared region in particular, this limitation is even more constraining, because the number of molecules that exist in this wavelength region is already small.        if inhibition of the fluorescence by the sugar is not complete, the detection sensitivity of the system is poor. To remedy this problem, certain manufacturers propose substrates in which the fluorophore group is bound to 2 saccharidic units by anomeric position 1. An example of this type of substrate is FDG. The initial inhibition of fluorescence is effectively increased by doubling the number of saccharidic units bonded to the fluorophore group. Nevertheless, the liberation of the fluorophore group and therefore of the fluorescence then also necessitates two enzymatic cleavages instead of one, and the detection sensitivity is therefore improved only slightly in such a system.        
The only commercially available enzymatic substrate that absorbs and emits in the near infrared is the substrate DDAOG, which is a conjugate of β-galactoside (G) and of 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO), used for detection of β-gal activity and sold by the Molecular Probes Co. (USA). This substrate absorbs at 645 nm and emits at 660 nm. Its mode of functioning, as described by Tung C.-H. et al., Cancer Research, 2004, 64, 1579-1583, is represented in Scheme B below:

However, even though this particular substrate absorbs and emits in the near infrared, it also exhibits a certain number of disadvantages:                its fluorescence is not completely inhibited when the fluorophore group is bonded to the saccharidic unit, which produces an initially non-negligible background noise and lowers the detection sensitivity. Thus, in the in vivo experiment described in the aforesaid article of Tung C.-H. et al., it can be seen that the injected DDAOG dose (0.5 mg) and the necessary exposure time (2 minutes) are very large compared with the traditional amounts and exposure times for applications of this type (generally 10-50 μg of substrate injected for an exposure time of 20 to 100 ms);        the absorption and emission spectra of the DDAO are very narrow and very close to one another, thus necessitating very good optical filtering to detect the signal relative to the initial background noise;        the absorption and emission spectra of the DDAO are still not shifted far enough into the red to be in an optimal optical window for performing in vivo imaging.        
There have also been proposed, especially in French Patent Application 2888938, fluorescent substrates having a saccharidic skeleton that carries, on the same saccharidic unit, a fluorophore group on the one hand and a group that inhibits the fluorescence of the fluorophore group on the other hand, its being understood that one of these two groups occupies the anomeric position of the saccharidic unit on which both groups are fixed, the other group occupying any other position whatsoever of the saccharidic unit.
Such fluorescent substrates permit the fixation of a greater variety of fluorophore groups that absorb and emit in the near infrared and therefore can be used in vivo. However, they also are not completely satisfactory, in particular because the affinity of enzymes for such substrates is greatly reduced and makes the process too inefficient.
Finally, there has very recently been developed a saccharidic sensor known as Gal-2SBPO, which results from conjugation of the substrate of β-galactosidase (β-D-galactopyranoside: Gal) and of a fluorescent water-soluble dye, the perchlorate of 9-di-3-disulfonyl propylaminobenzo[a]phenoxazonium (2SBPO) via a spacer arm that includes a peptide. In this enzymatic substrate (Gal-2SBPO), the fluorescence is inhibited by the peptide (glycine) included in the spacer arm connecting the saccharidic unit Gal and the fluorophore group 2SBPO proper (HO, N.-H. et al., Chem. Bio. Chem., 2007, 8, 560-566). The enzymatic activity of β-galactosidase induces cleavage between the sugar and the spacer arm, and the fluorophore group can then fluoresce after a final hydrolysis that separates it from its inhibitor peptide. The mode of functioning of this enzymatic sensor can be represented by the following Scheme C:

However, such a system still exhibits a certain number of limitations:                the choice of fluorophore is restricted solely to 9-di-3-disulfonyl propylaminobenzo[a]phenoxazonium perchlorate, which at present seems to be the only fluorophore group whose fluorescence can be inhibited by the presence of an amino acid such as glycine (HO, N.-H. et al., Tetrahedron, 2006, 62, 578-585).        the fluorescence intensity after the enzymatic activity is increased by a factor of only 7, which, given the current state of the capabilities of measuring instruments, still represents a small ratio for envisioning the use of this fluorescent marker for in vivo imaging.        
It is therefore to remedy all of these problems that the inventors have developed that which is the object of the invention.
The inventors effectively made it their objective to provide a fluorescent enzymatic substrate of saccharidic nature that does not have the disadvantage of prior art substrates and that in particular is very suitable, when so desired, for use in detection of enzymatic activities in vivo.